Project Summary Sphingolipids regulate a multitude of cellular and physiological processes and dysregulation of sphingolipid signaling is associated with cardiovascular and neurodegenerative disorders, and stroke. The long-term goal of the proposed research is to identify the cellular and molecular mechanisms by which sphingosine- 1-phospate (S1P), generated by sphingosine kinase, regulates the activation of the mitochondrial unfolded protein response (UPRmt). The UPRmt is critical for maintaining mitochondrial protein homeostasis in response to mitochondrial stress, but molecular mechanisms underlying its activation are not fully understood. My laboratory uses the model C. elegans to study the role of sphingolipid signaling in regulating neuronal function and neurotransmitter release. We recently uncovered a novel function for the sole sphingosine kinase ortholog, SPHK-1, in activating the UPRmt and in promoting organism-wide protection in response to a broad array of mitochondrial stressors. We found that SPHK-1 associates with mitochondria and that this association is regulated by mitochondrial stress generated either cell autonomously (in the intestine) or cell non-autonomously (by the nervous system). Here we seek to uncover the cellular and molecular mechanisms by which sphingolipid signaling is regulated during the UPRmt, how it activates the UPRmt and how it impacts mitochondrial homeostasis and survival in multicellular organisms. In Aim 1, we determine the molecular mechanism by which mitochondrial stress regulates SPHK-1 targeting and function. In Aim 2, we determine how neuroendocrine signaling regulates sphingolipid signaling during the UPRmt. In Aim 3, we identify cellular targets of S1P during the UPRmt and how they regulate transcriptional responses to ensure proper cellular homeostasis and survival in the face of stress. This proposal will advance mechanistic understanding of how mitochondrial sphingosine kinase is regulated and signals in vivo, and will further understanding of the mechanism underlying activation of the UPRmt.